Analyte detection with magnetic sensors

ABSTRACT

Methods for analyte detection with magnetic sensors are provided. Aspects of the methods include producing a magnetic sensor device having a magnetically labeled analyte from a sample, such as a serum sample, bound to a surface of a magnetic sensor thereof; and obtaining a signal, e.g., a real-time signal, from the magnetic sensor to determine whether the analyte is present in the sample. Also provided are devices, systems and kits that find use in practicing the methods of the invention. The methods, devices, systems and kits of the invention find use in a variety of different applications, including detection of biomarkers, such as disease markers.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e), this application is related to U.S.Provisional Application Ser. Nos. 60/973,973 filed Sep. 20, 2007 and61/086,411 filed Aug. 5, 2008; the disclosures of which are hereinincorporated by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos.1U54CA119367-01, PO1-HG000205, N43C0-2007-00030, and R43AI072800 awardedby the NIH, DARPA/Navy Grant No. N00014-02-1-0807, and U.S. Departmentof Defense grant number HDTRA1-07-1-0030-P-1. The Government has certainrights in this invention.

INTRODUCTION

A consensus is emerging that early detection and personalized treatmentin clinics based on genetic and proteomic profiles of perhaps 4-20biomarkers are the key to improving the survival rate of patients withcomplex diseases such as cancer. While the tools for large scalebiomarker discovery with hundreds to thousands of biomarkers areavailable, there are few biomolecular detection tools capable ofmultiplex and sensitive detection of biomarkers which can be readilyadopted in clinical settings for biomarker validation and forpersonalized diagnosis and treatment.

SUMMARY

Methods for analyte detection with magnetic sensors are provided.Aspects of the methods include producing a magnetic sensor device havinga magnetically labeled analyte from a sample, such as a serum sample,bound to a surface of a magnetic sensor thereof; and obtaining a signal,e.g., a real-time signal, from the magnetic sensor to determine whetherthe analyte is present in the sample. In certain embodiments, themethods include simultaneously quantifying one or more analytes in thesample. Also provided are devices, systems and kits that find use inpracticing the methods of the invention. The methods, devices, systemsand kits of the invention find use in a variety of differentapplications, including detection of biomarkers, such as diseasemarkers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of a magnetic nanotag-based protein detectionassay.

FIG. 2 shows graphs from the results of multiplex MNT-based assays.

FIG. 3 shows the results of a quadplex protein assay.

FIG. 4 shows a magnetic nanotag-based protein assay chip.

FIG. 5 shows an SEM image of magnetic nanotags.

FIG. 6 shows a schematic of a direct binding anti-IFN-γ assay in PBS.

FIG. 7 shows a test of sensor geometry and its effect on sensitivity.FIG. 7 a shows wide sensor vs. narrow sensor geometry. FIG. 7 b shows agraph of signal development vs. sensor size.

FIG. 8 shows a graph of a multiplex protein assay with nanotagamplification.

FIG. 9 shows a schematic and image of a reverse phase assay chip.

FIG. 9 a shows a schematic of reverse phase protein chip in whichanalytes are spotted on the chip directly and detection antibodies arethen incubated with the spots site-specifically. FIG. 9 b shows an imageof 64 samples spotted on a magneto-nano chip.

FIG. 10 shows magnetic signals of positive and control sensors versustime in a reverse phase protein detection experiment.

DEFINITIONS

The term “probe,” as used herein, refers to a moiety that iscomplementary to a target analyte of interest. In certain cases,detection of a target analyte requires association of a probe to atarget. In certain embodiments, a probe may be immobilized on a surfaceof a substrate, where the substrate can have a variety ofconfigurations, such as, but not limited to, a sheet, bead, or otherstructure. In certain embodiments, a probe may be present on a surfaceof a planar support, e.g. in the form of an array.

An “array,” includes any two-dimensional or substantiallytwo-dimensional (as well as a three-dimensional) arrangement ofaddressable regions, e.g. addressable regions, e.g., spatiallyaddressable regions or optically addressable regions bearing probes,particularly probes that are specific for the target analytes ofinterest. Where the arrays are arrays of nucleic acids or proteins, thenucleic acids or proteins may be adsorbed, physisorbed, chemisorbed, orcovalently attached to the arrays at any point or points along thenucleic acid chain.

An array is “addressable” when it has multiple regions of differentmoieties (e.g. different probes) such that a region (i.e., a “feature”or “spot” of the array) at a particular predetermined location (i.e., an“address”) on the array contains a particular probe. Array features aretypically, but need not be, separated by intervening spaces. An array isalso “addressable” if the features of the array each have a detectablesignature (e.g. a magnetic signature) that identifies the moiety presentat that feature.

The term “nucleic acid” as used herein describes a polymer of anylength, e.g., greater than about 2 bases, greater than about 10 bases,greater than about 100 bases, greater than about 500 bases, greater than1000 bases, up to about 10,000 or more bases composed of nucleotides,e.g., deoxyribonucleotides or ribonucleotides, and may be producedenzymatically or synthetically (e.g., PNA as described in U.S. Pat. No.5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions.Naturally-occurring nucleotides include guanine, cytosine, adenine andthymine (G, C, A and T, respectively).

The term “biomarker” as used herein refers to an indicator of aparticular disease state or a particular state of an organism. In somecases, a biomarker is a characteristic that is measured and evaluated asan indicator of normal biologic processes, pathogenic processes, orpharmacologic responses to a therapeutic intervention. In some cases, abiomarker may be a physiological indicator such as, but not limited to,blood pressure, heart rate or the like. In some cases, a biomarker maybe a molecular biomarker, such as but not limited to proteins, nucleicacids, carbohydrates, small molecules, and the like. For instance,examples of molecular biomarkers include, but are not limited to,elevated prostate specific antigen, which may be used as a molecularbiomarker for prostate cancer, or using enzyme assays as liver functiontests. In certain embodiments, a biomarker may indicate a change inexpression or state of a protein that correlates with the risk orprogression of a disease, or with the susceptibility of the disease to agiven treatment.

In certain cases, the disease may be a proliferative disease. As usedherein the term “proliferative disease” refers to a diseasecharacterized by the growth or rapid increase in size or number oftissues or cells. Examples include, but are not limited to, cancers,tumors, papillomas, sarcomas, carcinomas, and the like.

As used herein, the terms “processor”, “central processing unit”, or“CPU” refer to the part of a computer (i.e., a microprocessor chip) thatperforms data processing. In certain embodiments, a processor may beunder the control of a software program and thus is suitably programmedto execute all of the steps or functions required of it, and may alsoinclude any hardware or software combination that will perform suchrequired functions. For example, in some cases, a processor may inputdata (e.g. a data signal), process the data to obtain a result, andoutput the result on a computer-readable medium in a user-readableformat. In certain embodiments, the processor may be configured toobtain a real-time signal. As used herein, the term “real time” refersto events or signals that are detected or obtained as they happen or assoon as possible after they occur.

DETAILED DESCRIPTION

Methods for analyte detection with magnetic sensors are provided.Aspects of the methods include producing a magnetic sensor device havinga magnetically labeled analyte from a sample, such as a serum sample,bound to a surface of a magnetic sensor thereof; and obtaining a signal,e.g., a real-time signal, from the magnetic sensor to determine whetherthe analyte is present in the sample and, in certain embodiments, tosimultaneously quantify one or more analytes in the sample. Alsoprovided are devices, systems and kits that find use in practicing themethods of the invention. The methods, devices, systems and kits of theinvention find use in a variety of different applications, includingdetection of biomarkers, such as disease markers.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing embodiments of the invention, aspects of themethods will be described first in greater detail. Next, embodiments ofsystems and kits that may be used in practicing methods of invention arereviewed. In addition, a review is provided of embodiments of differentapplications in which methods, systems and kits of the invention finduse.

Methods

As summarized above, embodiments of the invention are directed tomethods of determining whether an analyte is present in a sample, i.e.,determining the presence or absence of an analyte in a sample, and, incertain embodiments, simultaneously quantifying one or more analytes inthe sample. Aspects of the methods include a step of producing amagnetic sensor device having a magnetically labeled analyte bound to asurface of a magnetic sensor thereof. Magnetic sensor devices that finduse in methods of the invention are described in greater detail below.The magnetically labeled analyte bound to a surface of a sensor of thedevice may be produced using a number of different protocols. Forexample, analyte may first be bound to a specific receptor on the sensorsurface, and then subsequently magnetically labeled. Alternatively,sample and magnetic label may be combined prior to contact with thesensor, and the resultant labeled analyte then allowed to bind to thesensor. In yet other embodiments, the sample of interest is firstpositioned on the sensor, and then contacted with magnetically labeledreagent specific for sample analyte of interest.

In certain embodiments, the methodology employed is one that uses a“real-time” signal. In certain embodiments, the assay of interest is amultiplex protein assay in which a complex sample, such as a serumsample, is assayed to determine whether two or more distinct proteinanalytes are present in the sample. In this latter type of embodiment,the signal employed may or may not be a real-time signal.

Real-Time Signal

In certain embodiments, the methods disclosed herein employ a “realtime” signal. As such, embodiments of the invention including obtaininga real time signal from the device and employing that signal todetermine at least the presence or absence of a given analyte ofinterest in a sample. Accordingly, embodiments of the invention observethe evolution in real time of the signal associated with the presence ofthe analyte of interest, and indeed of multiple analytes, as the targetlabeling evolves. The real time signal is made up of two or more datapoints obtained over a given period of time of interest, where incertain embodiments the signal obtained is a continuous set of datapoints obtained continuously over a given period of time of interest.

In some embodiments, the signal is observed while the assay system is inthe “wet” condition, that is, with the solution containing all theunknowns still in contact with the assay detection system. As such,there is no need to wash away all of the non-binding or irrelevantmolecules. This “wet” detection is possible because the magnetic fieldgenerated by the magnetic tag nanoparticle (e.g., 100 nm or less asdescribed elsewhere) decreases rapidly as the distance from thenanoparticle increases. Therefore, the magnetic field at the sensor ofthe nanoparticle bound to the captured target molecule exceeds themagnetic field from the unbound magnetic nanoparticles in the solution,which are both at a greater distance from the detector and are inBrownian motion. The term “proximity detection” as used herein refers tothis dominance at the sensor of the bound nanoparticles. Under the“proximity detection” scheme specifically absorbed analyte-nanotagconjugates at the sensor surface can be quantified without washing offthe nonspecific magnetic nanotags in the solution. Furthermore, incertain embodiments, with appropriate surface chemistry and captureprobes, the same magneto-nano biosensors for either protein or nucleicacid assays can be employed.

The magnetic nanotag binding kinetics can be approximately described bythe following equation:

${n = {\frac{n_{o}\left( {^{k_{on}C_{o}t} - 1} \right)}{^{k_{on}C_{o}t}} = {n_{0}\left( {1 - ^{{- k_{on}}C_{0}t}} \right)}}},$

where n (cm⁻²) is the density of captured streptavidin-coated nanotags,n_(o) is the original density of biotinylated linker antibodies on thesensor surface before the nanotag application, k_(on) (cm³s⁻¹) is theassociation rate constants for the specific absorption of nanotags onthe sensor surface, and C₀ is the nanotag concentration in the bulksolution away from the sensor surface. The initial slope of thereal-time signal trace at the moment of nanotag application is describedby the following equation:

${\frac{n}{t}_{t = 0}} = {k_{on}C_{0}{n_{0}.}}$

Since k_(on)C₀ is a constant for a given nanotag solution, the initialslope is directly proportional to the biotinylated linker antibodydensity n₀, which is in turn directly proportional to the analytedensity captured on the sensor surface. Therefore, in certainembodiments, instead of using voltage signal from magneto-nanosensors,the initial slope of the real-time signal trace can be used to quantifyanalyte concentrations. Furthermore, the slope of the real-time signaltrace at any time instant t can be derived as follows:

${\frac{n}{t}_{t = 0}} = {k_{on}C_{0}n_{0}{^{{- k_{on}}C_{0}t}.}}$

Therefore, the slope at an appropriate time t can be used to quantifyanalytes as long as the time instant t and constant k_(on)C₀ are keptthe same for standard curves and real assays.

In certain cases, the magnetic nanoparticles in magnetic assays canprecipitate, especially when the particle sizes are greater than about100 nm or when the biofunctionalization of the nanotags is not robust.In these cases, nanotag kinetics can be described as follows:

n=n ₀(1−e ^(−k) ^(on) ^(C) ⁰ ^(t))+k _(ns) C ₀ t,

where k_(ns) is a constant characterizing the nonspecific absorption ofmagnetic nanotags due to precipitation. In certain embodiments, readingsin the presence of precipitated nanoparticles can be achieved bysimultaneously recording the real-time signal traces of a probe sensor(positive sensor) and a control sensor (negative sensor). In thesecases, the real-time signal traces of the matched pair of positive andnegative sensors have similar linear slopes due to nanotagprecipitation, which are essentially equal to a constant of k_(ns)C₀.Therefore, by subtracting the real-time signal trace of the positivesensor with that of the matched negative sensor, the specific bindingsignal trace, which is representative of the nanotag density (and thusanalyte density) captured on the sensor surface, can be obtained.

The precipitation of nanoparticles is often caused by field introducedattraction between magnetic nanoparticles. In certain embodiments, theprecipitation of nanoparticles can be reduced by improved surfacemodification of magnetic nanoparticles or by using smaller fields duringassay to prevent aggregation of nanoparticles. If nanotags doprecipitate, they tend to settle to the bottom of the flow channel dueto gravity. Therefore, in certain embodiments, locating the sensors atthe top of a flow cell or a microfluidic channel which carries thesamples and nanotag solutions may help to reduce nonspecific signal dueto precipitation.

In certain embodiments, an indirect labeling method in magnetic assaysis used, i.e., nanotag labeling is done after the incubation of analyteswith the magneto-nano chip. In other embodiments, a direct labelingmethod may be used in which the analytes are first labeled with magneticnanotags and then incubated with the magneto-nano chip with immobilizedcapture probes. An aspect of both the indirect labeling method and thedirect labeling method is that the proximity detection capability of themagneto-nano sensors remain substantially the same. In embodiments thatemploy the direct labeling method, the real time signal traces containinformation about the binding kinetics of magnetic nanotag-analyteconjugates with capture probes.

Multiplex Protein Assay of Complex Samples

Aspects of the invention include the multiplex detection of the presenceor absence of proteins, nucleic acids, and other analytes of interest incomplex samples. For example, by “multiplex detection” is meant that twoor more distinct proteins that are different from each other and have adifferent amino acid sequence are detected, such as 4 or more, 6 ormore, 8 or more, etc., up to 20 or more, e.g., 50 or more, including 100or more, distinct proteins. As such, in some cases, the magnetic sensordevice may comprise two or more distinct magnetic sensors that eachspecifically detects a distinct analyte, such as four or more, 6 ormore, 8 or more, etc., up to 20 or more, e.g., 50 or more, including 100or more, distinct magnetic sensors. In certain embodiments, of interestis the multiplex detection of 2 to 20 distinct proteins, such as 4 to 20distinct proteins. Thus, in these embodiments, the magnetic sensordevice may comprise 2 to 20 distinct magnetic sensors that eachspecifically detects a distinct analyte, such as 4 to 20 distinctmagnetic sensors. In other cases, the magnetic sensor device maycomprise 20 or less distinct magnetic sensors that each specificallydetects a distinct analyte, such as 10 or less, including 4 or lessdistinct magnetic sensors.

By “complex sample” is meant a sample that may or may not have theproteins of interest, but also includes many different proteins andother molecules that are not of interest. In certain embodiments, thecomplex sample is a blood sample, by which is blood or a fractionthereof, e.g., serum. In certain embodiments, the complex sample is aserum sample. In certain embodiments, the complex sample assayed in themethods of the invention is one that includes 10 or more, such as 20 ormore, including 100 or more, e.g., 10³ or more, 10⁴ or more (such as15,000; 20,000 or even 25,000 or more) distinct (i.e., different)molecular entities, that differ from each other in terms of molecularstructure.

Systems

Systems of the invention are configured to practice the methods ofanalyte detection, and include magnetic sensor devices and nanotags.Magnetic sensor devices and nanotags employed in methods of theinvention may be those employed in any number of magnetic detectionsystems including those systems based on: spin valve detectors (alsoreferred to as spin valve film detectors), magnetic tunnel junction(MTJ) detectors, as well as those detectors described in U.S. patentapplication Ser. No. 10/829,505, the disclosure of which is hereinincorporated by reference.

In certain embodiments, the subject magnetic sensor device comprises asubstrate surface which displays magnetic sensors on the substratesurface. In some cases, the magnetic sensors have an analyte specificprobe that may be adsorbed, physisorbed, chemisorbed, or covalentlyattached to the magnetic sensors. In certain embodiments, the magneticsensor device comprises a substrate surface with an array of magneticsensors.

Any given substrate may carry one, two, four or more arrays disposed ona front surface of the substrate. Depending upon the use, any or all ofthe arrays may be the same or different from one another and each maycontain multiple distinct magnetic sensors. An array may contain one ormore, including two or more, four or more, 8 or more, 10 or more, 50 ormore, or 100 or more magnetic sensors. For example, 64 magnetic sensorscan be arranged into an 8×8 array. In certain embodiments, the magneticsensors can be arranged into an array with an area of less than 10 cm²or even less than 5 cm², e.g., less than about 1 cm², including lessthan about 50 mm², less than about 20 mm², such as less than about 10mm², or even smaller. For example, magnetic sensors may have dimensionsin the range of about 10 μm×10 μm to about 200 μm×200 μm, includingdimensions of about 100 μm×100 μm or less, such as about 90 μm×90 μm orless, for instance 50 μm×50 μm or less.

In certain embodiments, the magnetic sensor may comprise a plurality oflinear magnetoresistive segments, which are connected in series. Forinstance, the magnetic sensor can comprise 4 or more, such as 8 or more,including 16 or more, e.g. 32 or more, for example 64 or more, or 128 ormore linear magnetoresistive segments. The magnetoresistive segments caneach be about 5 μm wide or less, such as about 3 μm wide or less,including about 1.5 μm wide or less.

In certain embodiments, at least some, or all, of the magnetic sensorshave different analyte specific probes on their surface, such that eachanalyte specific probe displaying magnetic sensor each specificallydetects a distinct analyte. Areas in between the magnetic sensors may bepresent which do not carry any analyte specific probes. Suchinter-sensor areas, when present, could be of various sizes andconfigurations.

In certain embodiments, the substrate carrying the one or more arrayswill be shaped generally as a rectangular solid (although other shapesare possible), having a length of more than 4 mm and less than 150 mm,such as more than 4 mm and less than 80 mm, for instance less than 20mm; a width of more than 4 mm and less than 150 mm, such as less than 80mm, including less than 20 mm; and a thickness of more than 0.01 mm andless than 5.0 mm, such as more than 0.1 mm and less than 2 mm, includingmore than 0.2 mm and less than 1.5 mm, for instance more than about 0.8mm and less than about 1.2 mm.

Nanoparticles

Nanoparticles useful in the practice of embodiments the presentinvention are magnetic (e.g., ferromagnetic) colloidal materials andparticles. The magnetic nanoparticles can be high moment magneticnanoparticles which are small in size so as to be superparamagnetic, orsynthetic antiferromagnetic nanoparticles which contain at least twolayers of antiferromagnetically-coupled high moment ferromagnets. Bothtypes of nanoparticles appear “nonmagnetic” in the absence of a magneticfield, and do not normally agglomerate. In accordance with the presentinvention, magnetizable nanoparticles suitable for use comprise one ormore materials selected from the group consisting of paramagnetic,superparamagnetic, ferromagnetic, and ferrimagnetic materials, as wellas combinations thereof.

In certain embodiments, the magnetic nanoparticles possess the followingproperties: (1) their remnant magnetization is as small as possible sothat they will not agglomerate in solutions (either superparamagneticparticles or antiferromagnetic particles can satisfy this requirement);(2) the tags display high magnetic moments under a modest magnetic fieldof about 100 Oe so they can be readily detected; (3) the size of thetags may be comparable to the target biomolecules so that they do notinterfere with the binding interactions; (4) the tags are uniform andchemically stable in a biological environment; and/or (5) the tags arebiocompatible, i.e., water soluble and functionalized so that they arereadily attached to biomolecules of interest, e.g., a receptor thatspecifically binds to a target analyte.

In certain embodiments, the nanoparticles are high moment magneticnanoparticles such as Co, Fe or CoFe nanocrystals which aresuperparamagnetic at room temperature. They can be fabricated bychemical routes such as, but not limited to salt reduction or compounddecomposition in appropriate solutions. Examples of such magneticnanoparticles have been published in the literature (S. Sun, and C. B.Murray, J. Appl. Phys., 85: 4325 (1999); C. B. Murray, et al., MRSBulletin, 26: 985 (2001); S. Sun, H. Zeng, D. B. Robinson, S. Raoux, P.M. Rice, S. X. Wang, and G. Li, J. Am. Chem. Soc., 126, 273-279 (2004)).In certain embodiments, these particles can be synthesized withcontrolled size (e.g., about 5-12 nm), are monodisperse, and arestabilized with oleic acid. Magnetic nanoparticles and nanopowderssuitable for use with the present invention include, but are not limitedto Co, Co alloys, ferrites, cobalt nitride, cobalt oxide, Co—Pd, Co—Pt,iron, iron alloys, Fe—Au, Fe—Cr, Fe—N, Fe₃O₄, Fe—Pd, Fe—Pt, Fe—Zr—Nb—B,Mn—N, Nd—Fe—B, Nd—Fe—B—Nb—Cu, Ni, and Ni alloys. In other embodiments, athin layer of gold can be plated onto a magnetic core, or apoly-L-lysine coated glass surface can be attached to a magnetic core.Suitable nanoparticles are commercially available from, e.g.,Nanoprobes, Inc. (Northbrook, Ill.), and Reade Advanced Materials(Providence, R1).

In some cases, magnetic nanoparticle tags are fabricated by physicalmethods (W. Hu, R. J. Wilson, A. Koh, A. Fu, A. Z. Faranesh, C. M.Earhart, S. J. Osterfeld, S.-J. Han, L. Xu, S. Guccione, R. Sinclair,and S. X. Wang, Advanced Materials, 20, 1479-1483 (2008)) instead ofchemical routes, and are suitable for labeling the target biomoleculesto be detected. The tags comprise at least two thin ferromagneticlayers, preferably FexCoi.x, wherein x is 0.5 to 0.7, or FexCoi.x basedalloys. FexCoi_x has the highest saturation magnetization (about 24.5kGauss) among the known ferromagnetic materials (R. M. Bozorth,Ferromagnetism, D. Van Nostrand Company (1951)). These ferromagneticlayers are separated by nonmagnetic spacer layers such as Ru, Cr, Au,etc., or their alloys. In certain cases, the spacer layers areappropriately engineered to make the ferromagnetic layers coupledantiferromagnetically so that the net remnant magnetization of theresulting particles are zero or near zero. In certain embodiments, theantiferromagnetic coupling can be achieved via RKKY exchange interaction(see e.g., S. S. P. Parkin, et al., Phys. Rev. Lett., 64(19): 2304(1990)) and magnetostatic interaction (J. C. Slonczewski, et al., IEEETrans. Magn., 24(3): 2045 (1988)). In some cases, the antiferromagneticcoupling strength is moderate so that the particles can be saturated(i.e., magnetization of all layers become parallel) by an externalmagnetic field of about 100 Oe. In some cases, this can be achieved byadjusting layer thicknesses and by alloying the spacer layer.

In particular embodiments, to facilitate the bio-conjugation of thenanoparticle, a gold cap (or cap of functionally analogous or equivalentmaterial) is added at the top of the antiferromagnetic stack so that thenanoparticle can be conjugated to biomolecules via the gold-thiollinkage. Furthermore, appropriate surfactants can also be readilyimparted to the nanoparticles, rendering them water-soluble. The edge ofthe nanoparticles can also be passivated with Au or other thin inertlayers for chemical stability.

Any convenient protocol may be employed to fabricate the nanoparticlesdescribed above. For instance, in certain embodiments, a film stack canbe made of nanometer-scale ferromagnetic and spacer layers deposited onultrasmooth substrates (or a release layer). In some instances, a masklayer can be formed by imprinting, etching, self assembly, etc.Subsequently the mask layer and unwanted film stack are removed andcleaned off thoroughly. Then, the release layer is removed, lifting offnanoparticles which are the negative image of the mask layer. Theseparticles are eventually imparted with surfactants and biomolecules. Insome cases, the ultrasmooth substrate can be reused after thoroughcleaning and chemical mechanical polishing (CMP).

In other embodiments, the nanoparticles are fabricated with asubtractive fabrication method. In this case, the film stack is directlydeposited on the release layer followed by a mask layer. The film stackis etched through the mask layer, and eventually released from thesubstrate. These nanoparticles result from a positive image of the masklayer as opposed to the case in the additive fabrication method.

In certain embodiments, the size of the magnetic nanoparticles suitablefor use with the present invention is comparable to the size of thetarget biomolecule to be worked with, such that the nanoparticles do notinterfere with biological processes such as DNA hybridization.Consequently, the size of the magnetic nanoparticles is, in someembodiments, from about 5 nm to about 250 nm (mean diameter), such asfrom about 5 nm to about 150 nm, including from about 5 nm to about 20nm. For example, magnetic nanoparticles having a mean diameter of 5 nm,6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm,17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm,and 150 nm, as well as nanoparticles having mean diameters in rangesbetween any two of these values, are suitable for use with the presentinvention. Further, in addition to a spherical shape, magneticnanoparticles suitable for use with the present invention can be disks,rods, coils, or fibers.

In certain embodiments, synthetic antiferromagnetic nanoparticles may belarger than ordinary ferromagnetic particles. This is because, toprevent clumping, the nanoparticle must have no net magnetic moment (ora very small magnetic moment) in zero applied field. Antiferromagneticparticles may have zero magnetic moment in zero field at all sizes. Incontrast, for a ferromagnetic particle, its size may be below the“superparamagnetic limit”, which is, in some cases, about 20 nm or less,such as about 15 nm or less, including about 10 nm or less.

In certain embodiments, the synthetic nanoparticles described above canbe produced in large quantities using a large wafer and standard vacuumthin film deposition processes. For example, with a 6-inch round wafer,30-nm diameter nanoparticles at a rate of roughly 5×10¹² particles perrun can be produced, assuming each particle occupies a square of 60 nmby 60 nm on the wafer.

High Sensitivity Spin Valve Detectors

A spin valve detector is a metallic multilayer thin-film structure oftwo ferromagnetic layers spaced by a non-magnetic layer such as copper.One ferromagnetic layer, called the pinned layer, has its magnetizationpinned to a certain direction, while the magnetization of the otherferromagnetic layer, called the free layer, can rotate freely under anapplied magnetic field. The electrical resistance of a spin valvedepends on the relative orientation of magnetization of the free layerto that of the pinned layer. When the two magnetizations are parallel,the resistance is the lowest; when antiparallel, the resistance is thehighest. The relative change of resistance is called themagnetoresistance (MR) ratio. In some cases, the MR ratio of a spinvalve can reach more than about 10% in a small magnetic field, e.g.,about 100 Oe. Therefore, a spin valve can function as a sense elementfor the detection of a small magnetic particle that is attached to a DNAfragment as a label and immobilized onto the sensor surface. Since theparticle is magnetic (under a DC bias field or AC tickling field), itgenerates a magnetic field. The magnetic field may then affect theorientation of the free layer magnetization, causing a change in theelectrical resistance of the spin valve.

In some embodiments, the operation of a spin valve detector is describedas follows: 1) The magnetic nanoparticle under a DC bias field (Hb)generates a magnetic field around it. 2) The magnetic field will affectthe resistance of a spin valve closely underneath it. 3) Application ofan AC tickling field (Ht) will force the moment of particle tooscillate, resulting in an oscillating MR signal from the spin valve. Insome embodiments, in the in-plane mode, the spin valve detector signaldue to the magnetic nanoparticle has the same frequency f as the ACtickling field Ht, while in the vertical mode the signal has twice thefrequency of Ht. 4) A lock-in amplifier or frequency spectrum analyzeris used to pick up the oscillating signal with a high signal-to-noiseratio.

In certain embodiments, spin valves have a magnetoresistive (MR) ratioof about 1% to about 20%, such as about 3% to about 15%, including about5% to about 12%. Therefore, in certain embodiments, spin vales candetect a single magnetic nanoparticle of about 10 nm size in a narrowbandwidth (i.e., about 1 Hz or less) or with lock-in detection. In thesecases, by narrowing the noise bandwidth, a sufficient signal to noiseratio (SNR) is achieved even for single nanoparticle detection.

Spin valve detection may be performed with the in-plane mode (see e.g.,Li, et al., J. Appl. Phys. Vol. 93 (10): 7557 (2003)). In otherembodiments, the vertical mode, even though giving a slightly smallersignal amplitude, can be used when the electromagnetic interference(EMI) signal due to the AC tickling field in the detection system issignificant. The EMI signal tends to center at the frequency f of the ACtickling field, so it can be eliminated or greatly reduced if byperforming lock-in detection at the frequency 2f. Furthermore, in someinstances, a 2-bridge circuit can be used to eliminate any remainingEMI. Even more sophisticated signal acquisition and processing methodswith an AC modulation sense current and an AC tickling field at twodifferent frequencies have been published (e.g., S-J Han, H. Yu, B.Murmann, N. Pourmand, and S. X. Wang, IEEE International Solid-StateCircuits Conference (ISSCC) Dig. Tech. Papers, San Francisco Marriott,Calif., USA, Feb. 11-15, 2007.)

In certain embodiments, the signal from the spin valve detector due tothe magnetic tag depends on the distance between the magnetic tags andthe free layer of the spin valve, in addition to the geometry and biasfield of the spin valve itself. The detector voltage signal from asingle magnetic particle decreases with increasing distance from thecenter of the particle to the midplane of the spin valve free layer.

In certain embodiments, the free layer in the spin valves is on top ofthe pinned layer to facilitate detection of the magnetic nanoparticlesbecause the sensing magnetic field from a magnetic particle dropsmonotonically with the distance between the sensor and the particle.Furthermore, minimization of the distance between the magnetic particleand the top surface of the free layer, including the thickness of thepassivation layer protecting the spin valves facilitates magneticparticle detection.

In some instances, during operation of the detector array, a solution ofDNA (or protein) is flowed over the sensor surface to allow for affinitybinding of analytes of interest with corresponding capture agents on thesensor. Therefore, corrosion of the sensor surface is a concern becausedegradation of the detector surface could reduce sensitivity by reducingthe signal from biological binding events or by degrading the detectorsthemselves.

In certain embodiments, to reduce the corrosion of the sensor surface,magnetic detection schemes may include the addition of relatively thickpassivation layers to the detector surfaces. A trade-off occurs betweenretaining high sensitivity while sufficiently guarding againstdegradation. Thus, in certain embodiments, the detector combines anultrathin (i.e., about 10 nm or less) layer of passivation and verysmall magnetic nanoparticle tags (i.e., with a mean diameter of about 20nm or smaller), thus achieving a particle-center-to-detector distance ofless than about 30 nm (including the intervening DNA fragment length ofabout 10 nm), which is close enough to provide the necessary sensitivityfor single-tag detection. In certain embodiments, the ultrathin layersof passivation (such as Ta, Au, or oxide) suitable for use with thepresently disclosed detectors can have a thickness from about 1 nm toabout 10 nm, such as from about 1 nm to about 5 nm, including from about1 nm to about 3 nm. In certain embodiments, the ultrathin layers ofpassivation (such as Ta, Au, or oxide) suitable for use with thepresently disclosed detectors can have a thickness from about 10 nm toabout 50 nm, such as from about 20 nm to about 40 nm, including fromabout 25 nm to about 35 nm.

High Sensitivity Magnetic Tunnel Junction (MTJ) Detectors

An MTJ detector is constructed similarly to a spin valve detector exceptthat the non-magnetic spacer is replaced with a thin insulating tunnelbarrier such as alumina or MgO and that the sense current flowsperpendicular to the film plane. Electron tunneling between twoferromagnetic electrodes is controlled by the relative magnetization ofthe two ferromagnetic electrodes, i.e., tunneling current is high whenthey are parallel and low when antiparallel. In certain embodiments, theMTJ detector is composed of a bottom electrode, magnetic multilayersincluding a tunnel barrier, and a top electrode. In some cases, MTJdetectors have magnetoresistance ratios exceeding 200% (S. Ikeda, J.Hayakawa, Y. M. Lee, F. Matsukura, Y. Ohno, T. Hanyu, and H. Ohno, IEEETRANSACTIONS ON ELECTRON DEVICES, VOL. 54, NO. 5, 991-1001 (2007)) andinherently large device resistances, yielding higher output voltagesignals.

In certain embodiments, the MJT detector has a double-layer topelectrode. The first layer can be a thin gold layer (about 10 nm orless), which facilitates binding DNA or protein capture probes. Thesecond layer can be aluminum, copper or other conductive metals which donot bind with biomolecular probes, including but not limited topalladium, palladium alloys, palladium oxides, platinum, platinumalloys, platinum oxides, ruthenium, ruthenium alloys, ruthenium oxides,silver, silver alloys, silver oxides, tin, tin alloys, tin oxides,titanium, titanium alloys, titanium oxides, and combinations thereof. Insome instances, an aperture in the second layer, slightly smaller insize than the MTJ, is created either by a lift-off process or by etchinga uniform second layer. In these embodiments, the distance between thenanoparticle tag and the top surface of the free magnetic layer canrange from about 6 nm to about 100 nm, such as about 6 nm to about 40nm, including from about 6 nm to about 30 nm, such as from about 6 nm toabout 20 nm, including from about 6 nm to about 10 nm. Furthermore, thisarrangement may facilitate the reduction or prevention of currentcrowding (see e.g., van de Veerdonk, R. J. M., et al., Appl. Phys.Lett., 71: 2839 (1997)) within the top electrode which may occur if onlya very thin gold electrode is used.

Except that the sense current flows perpendicular to the film plane, theMTJ detector can operate similarly to the spin valve detector, eitherwith in-plane mode or vertical mode. As discussed above regarding spinvalve detectors, in certain embodiments, the vertical mode can be usedfor EMI rejection and, similarly, ultrathin passivation also applies toMTJ detectors. In addition, the first top electrode of thin gold on MTJdetectors can also serve the triple purposes of electrical conduction,ultrathin passivation, as well as specific biomolecular probeattachment.

In certain embodiments, at the same detector width and particle-detectordistance, MTJ detectors can give larger signals than spin valvedetectors. For example, for an MTJ detector with a junction area of 0.2μm by 0.2 μm and resistance-area product of 1 kOhm-μm², operating with aMR of 250% at a bias voltage of 250 mV, and Hb=35 Oe, Ht=100 Oe rms, thevoltage signal from a single 11 nm diameter Co nanoparticle whose centeris 35 nm away from the free layer midplane is about 200 μV, which insome instances is more than an order of magnitude larger than those forsimilar-sized spin valve detectors.

Utility

The subject methods, systems and kits find use in a variety of differentapplications where determination of the presence or absence, and/orquantification of one or more analytes in a sample is desired. Incertain embodiments, the methods are directed to detection of a set ofbiomarkers, e.g., 2 or more distinct protein biomarkers, in a sample.For example, the methods of invention may be used in the rapid, clinicaldetection of 2 or more disease biomarkers in a serum sample, e.g., asmay be employed in the diagnosis of a disease condition in a subject, inthe ongoing management or treatment of a disease condition in a subject,etc.

In certain embodiments, the subject methods, systems and kits find usein detecting biomarkers. In some cases, the subject methods, systems andkits may be used to detect the presence or absence of particularbiomarkers, as well as an increase or decrease in the concentration ofparticular biomarkers in blood, plasma, serum, or other bodily fluids orexcretions, such as but not limited to saliva, urine, cerebrospinalfluid, lacrimal fluid, perspiration, gastrointestinal fluid, amnioticfluid, mucosal fluid, pleural fluid, sebaceous oil, exhaled breath, andthe like.

The presence or absence of a biomarker or significant changes in theconcentration of a biomarker can be used to diagnose disease risk,presence of disease in an individual, or to tailor treatments for thedisease in an individual. For example, the presence of a particularbiomarker or panel of biomarkers may influence the choices of drugtreatment or administration regimes given to an individual. Inevaluating potential drug therapies, a biomarker may be used as asurrogate for a natural endpoint such as survival or irreversiblemorbidity. If a treatment alters the biomarker, which has a directconnection to improved health, the biomarker can serve as a surrogateendpoint for evaluating the clinical benefit of a particular treatmentor administration regime. Thus, personalized diagnosis and treatmentbased on the particular biomarkers or panel of biomarkers detected in anindividual are facilitated by the subject methods and systems.Furthermore, the early detection of biomarkers associated with diseasesis facilitated by the picomolar and/or femtomolar sensitivity of thesubject methods and systems. Due to the capability of detecting multiplebiomarkers on a single chip, combined with sensitivity, scalability, andease of use, the presently disclosed assay methods and systems finds usein portable and point-of-care or near-patient multiplexed moleculardiagnostics.

In certain embodiments, the subject methods, systems and kits find usein detecting biomarkers for a disease or disease state. In some cases,the disease is a cellular proliferative disease, such as but not limitedto, a cancer, a tumor, a papilloma, a sarcoma, or a carcinoma, and thelike. Thus, the subject methods, systems and kits find use in detectingthe presence of a disease, such as a cellular proliferative disease,such as a cancer, tumor, papilloma, sarcoma, carcinoma, or the like. Incertain instances, particular biomarkers of interest for detectingcancer or indicators of a cellular proliferative disease include, butare not limited to the following: C-reactive protein, which is anindicator of inflammation; transcription factors, such as p53, whichfacilitates cell cycle and apoptosis control; polyamine concentration,which is an indicator of actinic keratosis and squamous cell carcinoma;proliferating cell nuclear antigen (PCNA), which is a cell cycle relatedprotein expressed in the nucleus of cells that are in the proliferativegrowth phase; growth factors, such as IGF-I; growth factor bindingproteins, such as IGFBP-3; micro-RNAs, which are single-stranded RNAmolecules of about 21-23 nucleotides in length that regulate geneexpression; carbohydrate antigen CA19.9, which is a pancreatic and coloncancer biomarker; prostate specific membrane antigen, which is aprostate cancer biomarker; cyclin-dependent kinases; epithelial growthfactor (EGF); vascular endothelial growth factor (VEGF); proteintyrosine kinases; overexpression of estrogen receptor (ER) andprogesterone receptor (PR); and the like.

In certain embodiments, the subject methods, systems and kits find usein detecting biomarkers for an infectious disease or disease state. Insome cases, the biomarkers can be molecular biomarkers, such as but notlimited to proteins, nucleic acids, carbohydrates, small molecules, andthe like. Particular diseases or disease states that may be detected bythe subject methods, systems and kits include, but are not limited to,bacterial infections, viral infections, increased or decreased geneexpression, chromosomal abnormalities (e.g. deletions or insertions),and the like. For example, the subject methods, systems and kits can beused to detect gastrointestinal infections, such as but not limited to,aseptic meningitis, botulism, cholera, E. coli infection,hand-foot-mouth disease, helicobacter infection, hemorrhagicconjunctivitis, herpangina, myocaditis, paratyphoid fever, polio,shigellosis, typhoid fever, vibrio septicemia, viral diarrhea, etc. Inaddition, the subject methods, systems and kits can be used to detectrespiratory infections, such as but not limited to, adenovirusinfection, atypical pneumonia, avian influenza, bubonic plague,diphtheria, influenza, measles, meningococcal meningitis, mumps,parainfluenza, pertussis (i.e., whooping chough), pneumonia, pneumonicplague, respiratory syncytial virus infection, rubella, scarlet fever,septicemic plague, severe acute respiratory syndrome (SARS),tuberculosis, etc. In addition, the subject methods, systems and kitscan be used to detect neurological diseases, such as but not limited to,Creutzfeldt-Jakob disease, bovine spongiform encephalopathy (i.e., madcow disease), Parkinson's disease, Alzheimer's disease, rabies, etc. Inaddition, the subject methods, systems and kits can be used to detecturogenital diseases, such as but not limited to, AIDS, chancroid,Chlamydia, condyloma accuminata, genital herpes, gonorrhea,lymphogranuloma venereum, non-gonococcal urethritis, syphilis, etc. Inaddition, the subject methods, systems and kits can be used to detectviral hepatitis diseases, such as but not limited to, hepatitis A,hepatitis B, hepatitis C, hepatitis D, hepatitis E, etc. In addition,the subject methods, systems and kits can be used to detect hemorrhagicfever diseases, such as but not limited to, Ebola hemorrhagic fever,hemorrhagic fever with renal syndrome (HFRS), Lassa hemorrhagic fever,Marburg hemorrhagic fever, etc. In addition, the subject methods,systems and kits can be used to detect zoonosis diseases, such as butnot limited to, anthrax, avian influenza, brucellosis, Creutzfeldt-Jakobdisease, bovine spongiform encephalopathy (i.e., mad cow disease),enterovirulent E. coli infection, Japanese encephalitis, leptospirosis,Q fever, rabies, sever acute respiratory syndrome (SARS), etc. Inaddition, the subject methods, systems and kits can be used to detectarbovirus infections, such as but not limited to, Dengue hemorrhagicfever, Japanese encephalitis, tick-borne encephalitis, West Nile fever,Yellow fever, etc. In addition, the subject methods, systems and kitscan be used to detect antibiotics-resistance infections, such as but notlimited to, Acinetobacter baumannii, Candida albicans, Enterococci sp.,Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus,etc. In addition, the subject methods, systems and kits can be used todetect vector-borne infections, such as but not limited to, cat scratchdisease, endemic typhus, epidemic typhus, human ehrlichosis, Japanesespotted fever, louse-borne relapsing fever, Lyme disease, malaria,trench fever, Tsutsugamushi disease, etc.

Similarly, the subject methods, systems and kits can be used to detectcardiovascular diseases, central nervous diseases, kidney failures,diabetes, autoimmune diseases, and many other diseases.

Kits

Also provided are kits for practicing one or more embodiments of theabove-described methods. The subject kits may vary greatly, and mayinclude various devices and reagents. Reagents and devices of interestinclude those mentioned herein with respect to magnetic sensor devices,magnetic nanoparticles, binding agents, buffers, etc.

In certain embodiments, the subject kits include a magnetic sensordevice and a magnetic label. In these embodiments, the magnetic sensordevice comprises an analyte specific probe displaying magnetic sensorwhich displays a probe that specifically binds to an analyte on asurface thereof, and a processor configured to obtain a real-time signalfrom the magnetic sensor to determine whether the analyte is present ina sample. In some embodiments, the magnetic sensor comprises anultrathin passivation layer, where in certain cases the passivationlayer has a thickness of about 40 μm or less, such as about 30 μm orless, including about 20 μm or less. In further embodiments of thesubject kits, the sensor is a spin valve sensor, and in otherembodiments of the subject kits, the sensor is a magnetic tunneljunction sensor. In certain cases, the magnetic label of the subjectkits is a magnetic nonotag.

In certain embodiments, the subject kits include a magnetic sensordevice that comprises two or more distinct magnetic sensors that eachspecifically detects a distinct analyte, such as four or more, 6 ormore, 8 or more, etc., up to 20 or more, e.g., 50 or more, including 100or more, distinct magnetic sensors. Thus, the subject kits find use inthe multiplex detection of the presence or absence, and/orquantification of proteins, nucleic acids, or other analytes of interestin complex samples.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the Internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL I. Methods

A. Chip Fabrication—On silicon wafers with 150 μm thermal oxide, a spinvalve film with a layer sequence similar to that of hard disk drivesread heads was patterned by ion milling into individual sensors (S. X.Wang and G. Li, IEEE Trans. Magn., vol. 44, no. 7, 1687-1702 (2008)),each consisting of 32 linear segments of 1.5×100 μm connected in seriesand arranged to cover an area of 100×100 μm² (see FIG. 4). Each sensorhad a nominal resistance of 40 kΩ and a maximum magnetoresistance of12%. Corrosion resistant leads (Ta 5/Au 300/Ta 5) nm were sputterdeposited and patterned by lift-off. As suggested elsewhere (Schmitt, G.et al. Passivation and corrosion of microelectrode arrays. ElectrochimActa 44, 3865-3883 (1999)), the sensors were passivated with a tri-layeroxide (SiO₂ 10/Si₃N₄ 10 SiO₂ 10 nm), which was deposited at roomtemperature by ion beam sputter deposition. The leads were passivatedwith an additional tri-layer oxide (SiO₂ 100/Si₃N₄ 100/SiO₂ 100 nm). Atwo-component epoxy (EP5340, Eager Plastics, Chicago, Ill.) was used toassemble the chip and reagent well (Tygon® tubing, ¼″ ID×⅜″ OD, 6 mmlong) on the ceramic 84-pin chip carrier (LCC08423, SpectrumSemiconductor Materials, San Jose, Calif.). A layer of about 0.5 mm ofthe same epoxy was used to mask some of the sensors (see FIG. 4), so asto create two adjacent but separate sites for subsequentbiofunctionalization. The masked sensors, no longer able to detectnanotag binding, serve as electrical signal references (see section G,below).B. Surface Preparation—The assembled chips were thoroughly washed withacetone, methanol, isopropanol, and de-ionized water. A ten minute UVozone treatment (UVO Cleaner Model 42, Jelight, Irvine, Calif.) was usedto remove organic residues. To form the base layer of thebiofunctionalization, a 2% solution of polyethyleneimine (PEI, CAS9002-98-6, Sigma-Aldrich) in deionized water was applied to the chipsurface for 2 minutes. The chips were then rinsed with deionized waterand then baked at 150° C. for 5 minutes to solidify the adsorbed PEI.C. Muliplex Protein Assays—Up to four different probes were manuallydeposited in the form of 1 μL droplets, containing antibodies(Anti-IL-10, Anti-IL-1α, Anti-TNF-α, and Anti-VEGF) at a concentrationof 500 μg/mL, onto different areas of the chip to functionalize thesensor array. Additional control sensors were functionalized with a 1 μLdroplet of BSA 100 mg/mL. The sensor functionalizations were incubatedfor 30 minutes at 4° C. and 95% relative humidity. The chips were thenrinsed twice with blocking buffer (1% BSA, 0.2% Tween 20, in PBS) toblock any remaining non-specific surface binding sites. Samples wereprepared by diluting the protein analytes (TNF-α, 17.5 kDa; IL-10, 18.5kDa; IL-1α, 18 kDa) in either PBS buffer or in 50% human serum (balancePBS) to the desired concentrations. 100 μL of sample solution werepipetted into the reagent well of a chip and incubated for 1 hour atroom temperature. Subsequently, the chip was rinsed twice with blockingbuffer (0.1% BSA, 0.2% Tween 20, in PBS). A multiplex linker antibodysolution was prepared consisting of four biotinylated antibodies, onefor each potential analyte, at a concentration of 2 μg/mL in PBS. 100 μLof this linker antibody solution were incubated in the reagent well ofthe chip for 1 hour at room temperature. With the linker solution stillin place, the chips were then transferred over to the measuring stationfor MNT-based analyte quantification.D. hCG Assays—All chips were uniformly functionalized with a singlecapture probe, anti-human-hCG 1 mg/mL, incubated overnight at 4° C.,then rinsed twice with blocking buffer. Pure serum samples spiked withhCG to 2.5, 25, 250, and 2,500 IU/L were supplied by the U.S. NationalCancer Institute and diluted 1:1 with PBS buffer. Analyte incubationtime was 1 hour, and linker antibody, 5 μg/mL in PBS, was incubated for90 minutes. With the linker solution still in place, the chips were thentransferred over to the measuring station for MNT-based analytequantification. The concentrations of hCG after dilution with PBS havebeen converted using 1 IU/L=1.9 pM (Birken, S. et al. Preparation andcharacterization of new WHO Reference Reagents for human chorionicgonadotropin and metabolites. Clin. Chem. 49, 144-154 (2003); Sturgeon,C. M. & Ellis, A. R. Standardization of FSH, LH and hCG—Current positionand future prospects. Mol. Cell. Endocrinol. 260, 301-309 (2007)).E. MNT-Based Analyte Quantification—To remove the linker antibodysolution and to confirm signal baseline stability, the chips were rinsedwith MNT-free PBS buffer several times. While the associated wet/drytransitions did occasionally shift the baseline slightly, these shiftswere reversible and usually negligible compared to the signals ofinterest. The absolute signal level on contact with MNT-free buffer wastaken to be zero. The MNT-free buffer solution was then aspirated fromthe well and replaced with 50 μL of streptavidin-coated MNT stocksolution (MACS 130-048-102, Miltenyi Biotec). The nanotag solution wasincubated without stirring for the next 20 minutes at room temperature.The signal levels at the end of this 20 minute nanotag incubation timewere taken as the final result of the assay.F. Optional Nanotag Amplification—At the end of the initial 20 minuteMNT incubation period, the well was rinsed five times with PBS, and thenre-filled with 50 μL of the biotinylated linker antibody solution. Thissolution was incubated for five minutes, attaching biotinylatedantibodies to the already adsorbed MNTs. Since these linker antibodiescan have multiple biotin sites, new binding sites for additional MNTswere created this way on the existing MNTs. After five minutes, thesolution was aspirated, the well rinsed five times with PBS, and 50 μLof MNT stock solution were added, resulting in the generation of anadditional MNT binding signal. This amplified the signal levels by afactor of about 2× with each iteration.G. Electronics—An alternating current of 7 μA_(rms) at 500 Hz wasapplied to each sensor. An alternating in-plane tickling field of 80Oe_(rms) at 208 Hz was applied perpendicular to the sensor segments toestablish a magnetic signal baseline, which is minimally perturbed inthe vicinity of any nanotags. This perturbation of baseline signal isour net signal. A steady bias field of 50 Oe was also applied along thelongitudinal direction of the sensor segments to facilitate a coherent(low noise) rotation of the sensor's magnetic domains in response to the208 Hz tickling field. The signal level was measured by performing afast Fourier transform of the voltage across each sensor once every 2seconds and recording the magnitude of the (amplitude modulationgenerated) 708 Hz spectral component, which in this setup is primarily ameasure of the alternating magnetic tickling field strength in theimmediate vicinity of the sensor. To reduce the common mode and sensordrift, every functionalized sensor was measured differentially against areference sensor, i.e., against a sensor which was covered with a layerof epoxy thick enough to positively prevent any MNT detection.Additional details of this measurement setup are described elsewhere(Han, S. J., Xu, L., Wilson, R. J. & Wang, S. X. A novel zero-driftdetection method for highly sensitive GMR biochips. IEEE Trans. Magn.42, 3560-3562 (2006)).

II. Results & Discussion

In certain embodiments, magnetic nanotag (MNT)-based biomolecular assaysare capable of fast and sensitive multiplex protein detection involvingserum samples.

Micron-sized labels may diffuse slowly, are prone to magneticinteraction and subsequent precipitation, and are bulky compared tonanometer-sized analyte molecules. In contrast, in certain embodiments,nanometer sized MNTs may be used, such as commercially available 50 nmMACS MNTs, which have a very small magnetic signature but which exhibitlong term suspension stability and excellent binding selectivity. Insome cases, to enhance the sensitivity of the assay, the passivation ofthe SV sensors can be thinned to about 30 nm or less. The resultingsensitivity allows the reliable detection of minute magnetic signatures,such as from MACS MNTs, provided that they are in the immediateproximity of the sensor. By combining small magnetic moment MNTs withvery sensitive proximity detection, the sensors disclosed hereinprimarily detect MNTs that are bound to the sensor surface. If unboundMNTs are stable in suspension, then the signal contribution from theseextraneous unbound MNTs is negligible, and washing steps, which aretypically required to remove unbound signal-generating labels, can beomitted. In practice, this suppression of unbound labels means that thetrue amount of currently bound nanotags can be observed in real-time,and that negative control sensors experience no signal shift duringnanotag application and removal. Thus, in certain embodiments, simpleone-step homogeneous assays with no washing steps are provided, whichfind utility in clinical applications.

Methods for early cancer detection via quantification of cancer-relatedcytokines are also provided. Analytes to be detected in the MNT-basedprotein assay include, but are not limited to vascular endothelialgrowth factor (VEGF), tumor necrosis factor alpha (TNF-α),interleukin-10 (IL-10), and interleukin-1-alpha (IL-1α).

FIG. 1 shows a schematic of the detection scheme in which analyte iscaptured on the sensor surface and quantified with streptavidin-coatedMNTs. Probe sensors were functionalized with capture antibodies specificto the chosen analyte, while control sensors were blocked with a 10 wt %BSA solution (FIG. 1 a). During analyte incubation, the probe sensorscaptured a fraction of the analyte molecules (FIG. 1 b). A biotinylatedlinker antibody was subsequently incubated which binds to the capturedanalyte (FIG. 1 c), and which provides binding sites for thestreptavidin-coated magnetic nanotags. Streptavidin-coated magneticnanotags were then incubated (FIG. 1 d), and the nanotag binding signal,which saturates at an analyte concentration-dependent level, was used toquantify the analyte concentration.

In some cases, the analyte and linker antibody incubation time (FIGS. 1b, 1 c) is one hour or less, while the MNT-based quantification (FIG. 1d) requires less than fifteen minutes. In certain embodiments, the totalassay time can be reduced to below 30 minutes, for example throughanalyte incubation in a microfluidic channel, so that the methods maysuitable for a physician office laboratory or point-of-careapplications. In some cases, the MNT detection scheme can be carried outon a chip, which has an array of 64 SV sensors and a 200 μL reactionwell placed on top (see FIG. 4), so that the reagents can be pipettedand aspirated easily. In certain cases, the electronic instrumentationis able to record and display 16 sensor signals per chip in real-time,with an update rate of 0.5 Hz per sensor. In some cases, two of the 16measured sensors are covered with epoxy and used to record an electronicreference signal, so that 14 sensors per chip can be used to recordassay signals.

Actual binding curve (signal vs. time) data from MNT-based immunoassayswith multiple probes is shown in FIG. 2. The average signal ±1 S.D.(standard deviation) is shown. The time t=0 defines the moment of MNTnanotag application in the final step of the assay. Beginning at t=0,the signal rise reflects the binding of MNTs to the SV sensor surface inreal-time. As a result of the indirect labeling method used here, thereal-time data contains information about the MNT binding kineticsrather than the analyte binding kinetics, but the saturation level of anMNT binding curve is taken as a direct measure of binding site abundanceon the sensor surface, which in turn is determined by the concentrationof the previously applied analyte. In addition to analytequantification, the real-time MNT binding curves are also used toidentify and eliminate sources of error, as they help to distinguishproper (i.e., continuous, steady, saturating) from improper (i.e.,discontinuous, noisy, drifting) sensor operation. For example, in somecases, the signal quickly stabilizes despite an excess amount ofnanotags in the solution, which indicates that in the absence ofsuitable binding sites, the nanotags do not precipitate or bindspontaneously. This facilitates precise analyte quantification. The MNTbinding curves also give the time needed for reaching signal saturation,i.e., MNT binding equilibrium. In certain embodiments, the net signalgains at time t=15 minutes are reported to compare different assay runs,but when few MNT binding sites are available (i.e., due to low analyteconcentration), MNT quantification can reach equilibrium in as little as60 seconds.

An example of such rapid MNT quantification can be seen in FIG. 2 a. Inthis experiment (FIG. 2 a), fourteen sensors on a chip werefunctionalized as follows: 4 sensors anti-IL-1α, 8 sensors anti-TNF-α,and 2 sensors BSA. A sample consisting of 5.6 pM IL1-α and 0.6 pM TNF-αin PBS was then incubated for one hour. The resulting binding signalslevel off approximately one minute after MNT application, and the signallevels of 1.9 μV for TNF-α (8 sensors) and 4.1 μV for IL-1α (4 sensors)are significantly larger and distinct from the non-specific signal of0.6 μV on the BSA-functionalized control sensors.

In another experiment (FIG. 2 b), a chip was similarly functionalized: 5sensors anti-TNF-α, 2 sensors anti-IL-1α, and 2 sensors BSA. However, inthis experiment the sample consisted of 5.7 pM TNF-α in 50% serum,balance PBS. Both the sensors blocked with BSA and the IL-1α sensorswere negative controls. The resulting average signal saturated at 3.2 μVafter about two minutes. The negative controls, consisting of both theanti-IL-1α and BSA functionalized sensors, showed an average of 0.1 μV(range +0.3 μV to −0.3 μV), which indicates an average signal tobackground ratio of 32:1 at this concentration.

To demonstrate the signal vs. concentration scaling relationship of ananalyte in 50% serum over large changes in analyte concentration, aseries of MNT-based immunoassays were performed to detect humanchorionic gonadotropin (hCG) which was spiked into 50% serum. In thisexperiment, five chips were functionalized with anti-hCG and thenexposed to different concentrations of hCG in 50% serum. Fourteensensors were measured per chip (see FIG. 2 c). In this case, hCG waschosen as a model analyte because reference hCG samples, high qualityantibodies, and comparable commercial hCG assays are readily available.The results are shown in FIG. 2 c. The lowest hCG concentration, 2.4 pMin 50% serum, resulted in a 14-sensor median signal of 13.6 μV (max=27μV, min=8.2 μV, SD=4.6 μV). In addition, for each ten-fold increase inhCG concentration, the signal approximately doubled. The resultsdemonstrate the detection of hCG concentrations over at least fourorders of magnitude, down to the serum baseline level, which is about 1pM.

Performing the same assay and label amplification in analyte-free PBS(see FIG. 2 b control assay) shows that the control signal is in a rangeof about 2.5 μV_(rms) to about 3.5 μV_(rms) which is significantly lowerthan the signal expected from 1 pM hCG in serum. Extrapolation of thescaling trend down to the background level indicates that the MNT-basedassay can detect about 10 femtomolar concentrations of hCG in serum,which is more sensitive than commercial ELISA kits (i.e., with asensitivity of about 4 pM). In certain embodiments, higher signal levelscan be achieved by successively adsorbing two or more layers of nanotags(e.g. three layers of nanotags) by in situ nanotag amplification. Thenanotag amplification uniformly elevates all signal levels, includingthe control signal, thus nanotag amplification facilitates the detectionof signals which are initially too low to be precisely quantified. Insome cases, the relative signal vs. concentration scaling relationshipremained unchanged by nanotag amplification.

FIG. 3 shows the results of a multi-analyte, multi-probe assay performedon a single chip. In this experiment, four regions of the chip werefunctionalized, each with one of four capture antibodies (anti-TNF-α,anti-IL-10, anti-VEGF, and anti-IL-1α). The chip was then exposed to asubset of the four potentially recognized analytes, namely TNF-α58 pMand IL-10 at a concentration of 54 pM in PBS. Thus, the VEGF sensors andthe IL-1α sensors served as negative controls. Fourteen sensors weresubsequently chosen from the sensor array to measure the signals fromeach of the four regions with a certain redundancy (ranging fromreplicate to quadruple). The initial MNT quantification was enhancedwith one round of nanotag amplification. As seen in FIG. 3, only thesensors with matching analytes (in this case, TNF-α and IL-10) gavelarge signals as expected. Signal variation among sensors with identicalfunctionalization was small. In certain embodiments, the chip'ssensitivity appears to vary with the type of functionalization, sinceIL-10 was observed to give a larger signal than TNF-α at similarconcentrations. This indicates that, in certain cases, the analyteaffinity of each functionalization can be different. In some cases, thesmall but non-zero signals on the VEGF and IL-1α sensors may be due to asmall amount of cross-reactivity, which depends on the matching andquality of the assay antibodies.

In certain embodiments, with carefully screened antibodies, theMNT-based analyte quantification method can be used for clinicallyrelevant protein detection in real world serum samples, and multiplexprotein detection can be readily performed with this method. In somecases, up to 256 different probes, including up to 128, for instance upto 64 different probes can be accommodated and simultaneously measured.In some cases, as discussed above, the sensor can be a spin valve sensoror a magnetic tunnel junction sensor. In addition, as discussed in moredetail below, sensitivity can be increased by using sensors withnarrower segments (see FIG. 7). Furthermore, the analytic sensitivity ofMNT-based assays capture agents can be enhanced further with higheraffinity, and similarly small but higher magnetic moment MNTs. Due tothe capability of detecting multiple biomarkers on a single chip,combined with sensitivity, scalability, and ease of use, the presentlydisclosed protein assay method finds use in portable and point-of-careor near patient multiplexed molecular diagnostics. In addition, incertain embodiments, SV sensors are pH-insensitive, with no “bleaching”of MNTs and no magnetic background from bio-systems. In some cases,MNT-based assays produce signals that are stable even during changes ofexperimental conditions, such as wet to dry transitions. In otherembodiments, MNT-based analyte quantification can also be combined withmagnetic separation techniques. For example, analyte extraction and thefirst round of molecular amplification can be combined into a singlemagnetic separation step to achieve an ultra-sensitive proteindetection. Magnetic forces can also be used to draw MNT-labeled analytestowards the sensors, thereby reducing diffusion distances and assay timeand improving sensitivity.

FIG. 4 shows a magnetic nanotag-based protein assay chip. The chip has a200 μL reaction well and is supported by an 84-pin ceramic base (seeFIG. 4 a). Embedded in the bottom of the reaction well are 64 sensors inan 8×8 array (see FIG. 4 b). Each sensor has an active area of roughly90×90 μm² and consists of 32 linear magnetoresistive segments, each 1.5μm wide, which are connected in series (see FIG. 4 c). FIG. 4 d showsthe edge of one such sensor segment and bound nanotags imaged with ascanning electron microscope.

FIG. 5 shows an SEM image of magnetic nanotags. After the MNT-basedanalyte quantification, the MNT solution was aspirated and the chipswere rinsed twice with de-ionized water to remove salt residues whichotherwise would obscure bound MNTs. Chips were then metallized with 1 nmof AuPd by DC sputtering (Hummer V) to enhance image contrast. Imageswere obtained with an FEI Sirion XL30 scanning electron microscope.

The average diameter of the magnetic nanotags is about 50 nm(commercially available Miltenyi MACS 130-048-102). Upon metallizationand inspection in the SEM, these particles appear as roughly 35 nmirregular spheres as shown in FIG. 5, with frequent multi-particleclusters. MACS nanotags appear to contain only a small fraction ofmagnetic material dispersed throughout a non-magnetic organic matrix towhich the functionalization (streptavidin) is bound. In someembodiments, high coverage density was achieved by applying MACS stocksolution to a test chip that had been functionalized with biotinylatedbovine serum albumin (BSA).

FIG. 6 shows a schematic of a direct binding anti-IFN-γ assay in PBS. Inthis assay, the analyte is biotinylated anti-IFN-γ, which is captured byIFN-γ functionalized sensors and quantified with streptavidin-coatedmagnetic nanotags. In some cases, these chips exhibit a reversiblesignal baseline shift during wet-dry transitions, as can be seen in FIG.6 d from time −2<t<0 minutes. In certain embodiments, the reversiblebaseline shift can be reduced through passivation, as described above.

In FIG. 6 a, probe sensors were functionalized with a 1 μL droplet ofIFN-γ, 100 μg/mL in PBS buffer, for 30 minutes at 4° C. Control sensorswere functionalized with a 1 μL droplet of IL6-sR, 100 μg/mL in PBSbuffer. The functionalized chip was then rinsed with a 1% BSA in 1×PBSbuffer solution to block non-specific adsorption sites.

In FIG. 6 b, the chip well was filled with 100 μL of analyte, e.g.biotinylated anti-IFN-γ 670 pM (100 ng/mL) in PBS buffer. The analytewas incubated in the entire well of the chips for 1.5 hours at 30° C.The chips were then rinsed with 0.1% BSA in TPBS and transferred to themeasuring station for subsequent analyte quantification.

In FIG. 6 c, the chip well was filled with 100 μL of Miltenyi MACS stocksolution, and the developing MNT binding signal was recorded (see FIG. 6d). The MNT binding signals after 20 minutes of incubation were taken asa measure of analyte concentration (see FIG. 6 e). The small error barsindicate the electrical noise of the measurement, which was much smallerthan the variance of signals from identically functionalized sensors inthis experiment.

FIG. 7 shows a test of sensor geometry and its effect on sensitivity.FIG. 7 illustrates the evaluation of the effect of sensor segment widthwhile keeping other parameters of the sensors the same. FIG. 7 a shows aschematic illustration of simplified sensors with either a wide geometryor a narrow geometry. The schematic illustration on the left of FIG. 7 ashows a wide sensor with three segments, each 3 μm wide. The schematicillustration on the right of FIG. 7 a shows a narrow sensor with sixsegments, each 1.5 μm wide. Thus, the total sense current i, theresistance, the sensing area, and the sense current density of these twosensors can all be identical.

To ensure identical experimental conditions for both the wide and narrowsensor types, chips were fabricated that carried both sensor variantsinside the same reaction well. The entire reaction well was uniformlyfunctionalized with 100 μL of biotinylated BSA, 200 μg/mL, incubated for30 minutes at room temperature. No analyte was necessary, since the MNTscan bind directly to the biotinylated BSA. The chips were rinsed twicewith PBS and transferred to the measuring station for MNTquantification.

The results (see FIG. 7 b) indicate that patterning sensors more finely,as shown in the narrow sensor geometry on the right of FIG. 7 a, resultsin better sensitivity. The median curve for each type of sensor is shown(N=4 curves for each sensor type), with the error bars indicating ±1standard deviation (see FIG. 7 b). The signal from the 1.5 μm sensorswas consistently stronger than that from the 3.0 μm sensors (see FIG. 7b). The results show that using more finely patterned sensors withsubmicron line widths can facilitate an increase in sensitivity.

FIG. 8 shows a graph of a multiplex protein assay with nanotagamplification, which shows the effect of an optional magnetic nanotag(MNT) amplification step in a multiplex assay. A chip was functionalizedwith two probes (anti-TNF-α and anti-IL-1α) and one control (BSA), andthe applied sample contained 5.7 pM of TNF-α in PBS. The initialMNT-based analyte quantification was performed from 0<t<4.5 minutes.From 4.5<t<6.5 minutes, the chip was first washed, then brieflyincubated with biotinylated linker molecules, which attached to thealready adsorbed streptavidin-coated MNTs. The already adsorbed MNTswere thus able to capture additional MNTs. A second charge of MNTs wasthen incubated from t>6.5 minutes. As can be seen from the TNF-αsensors, additional MNTs were captured and a second binding curve wasobserved, which leveled off at almost twice the signal level of theoriginal MNT binding curve.

III. Reverse Phase

In reverse phase protein (PRP) microarrays, samples from differentpatients are spotted on a chip and then are incubated with detectionagents in a high throughput and multiplexed format. Magneto-nano chipsspotted with human cell lysate or serum samples were prepared and thenincubated with detection agents made up of selected biomarkers tomeasure human peripheral blood samples (see FIG. 9). A schematic of areverse phase protein (RPP) chip is shown in FIG. 9 a, and FIG. 9 bshows an image of 64 samples spotted on a magneto-nano chip. A graph ofthe results of protein assays detecting spotted EpCam antigens (a cancerbiomarker) using magnetic nanotag sensing is shown in FIG. 10.

In one embodiment, samples containing antigen (e.g., EpCam antigen) werespotted on selected sensors (positive sensors), while no antigen wasspotted on control sensors on the same chip. Then, the EpCam antibodyfunctionalized MACS nanoparticle solution was applied to the chip, andthe magnetic signals from the sensors were recorded in real time (seeFIG. 10), where MACS solution was applied slightly before time t=500seconds. By fitting the magnetic signal versus time curve, kineticconstants between EpCam antibody and antigen can be extracted. If thesolution is diluted at a certain time point, e.g., t=2500 second,desorption of EpCam antibody from antigen occurs, and the off-rate canbe extracted by fitting the magnetic signal versus time curve (data notshown).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A method of determining whether an analyte is present in a sample,said method comprising: producing a magnetic sensor device having amagnetically labeled analyte bound to a surface of a magnetic sensorthereof; and obtaining a real-time signal from said magnetic sensor todetermine whether said analyte is present in said sample.
 2. The methodaccording to claim 1, wherein said producing comprises: (a) providing amagnetic sensor device having an analyte specific probe displayingmagnetic sensor which displays a probe that specifically binds to saidanalyte on a surface thereof; and (b) contacting said analyte specificprobe displaying magnetic sensor with said sample to produce a sampleexposed sensor.
 3. The method according to claim 2, wherein said samplehas been magnetically labeled prior to said contacting step.
 4. Themethod according to claim 2, wherein said method comprises magneticallylabeling said sample exposed sensor after said contacting step (b). 5.The method according to claim 1, wherein said producing comprises: (a)positioning said sample on a said surface of said magnetic sensor toproduce a sample displaying sensor surface; and (b) magneticallylabeling said sample displaying sensor surface.
 6. The method accordingto claim 1, wherein said magnetic sensor device comprises two or moredistinct magnetic sensors that each specifically detects a distinctanalyte.
 7. The method according to claim 6, wherein said magneticsensor device comprises four or more distinct magnetic sensors that eachspecifically detects a distinct analyte.
 8. The method according toclaim 7, wherein said magnetic sensor device comprises 20 or lessdistinct magnetic sensors that each specifically detects a distinctanalyte.
 9. The method according to claim 1, wherein said magneticsensor is a spin valve sensor.
 10. The method according to claim 1,wherein said magnetic sensor is a magnetic tunnel junction sensor. 11.The method according to claim 1, wherein said analyte is a protein. 12.The method according to claim 1, wherein said analyte is a nucleic acid.13. The method according to claim 1, wherein said sample is a complexsample.
 14. The method according to claim 13, wherein said complexsample is a serum sample.
 15. A method of determining whether two ormore distinct protein analytes are present in a serum sample, saidmethod comprising: contacting said serum sample with a surface of amagnetic sensor device having, for each of said two or more distinctprotein analytes, an analyte specific probe displaying magnetic sensorwhich displays a probe that specifically binds to an analyte on asurface thereof; and obtaining a signal from each magnetic sensor ofsaid device to determine whether said two or more distinct proteinanalytes are present in said sample.
 16. The method according to claim15, wherein said signal is a real-time signal.
 17. The method accordingto claim 16, wherein said method comprises magnetically labeling saidsample after said sample is contacted with said sensor surface.
 18. Themethod according to claim 16, wherein said method comprises magneticallylabeling said sample before said sample is contacted with said sensorsurface. 19-25. (canceled)
 26. A magnetic sensor device comprising: ananalyte specific probe displaying magnetic sensor which displays a probethat specifically binds to an analyte on a surface thereof; and aprocessor configured to obtain a real-time signal from said magneticsensor to determine whether said analyte is present in a sample.
 27. Themagnetic sensor device according to claim 26, wherein said magneticsensor comprises an ultrathin passivation layer. 28-36. (canceled)
 37. Akit comprising: (a) a magnetic sensor device comprising: (ii) an analytespecific probe displaying magnetic sensor which displays a probe thatspecifically binds to an analyte on a surface thereof; and (ii) aprocessor configured to obtain a real-time signal from said magneticsensor to determine whether said analyte is present in a sample, and/orto quantify the said analyte; (b) a magnetic label. 38-42. (canceled)43. The method according to claim 4, wherein said magnetically labelingcomprises nanotag amplification.
 44. The method according to claim 43,wherein said nonatag amplification comprises successively absorbing twoor more layers of nanotags.
 45. The magnetic sensor device according toclaim 26, wherein said magnetic sensor comprises an ultrathinpassivation layer with a total thickness